The objective of this project is to analyze host-defense mechanisms against viral infection in the model organism Drosophila melanogaster, and to do so in the light of concurrent studies of viral infection in mice, carried out by our colleagues in La Jolla and in Osaka. By studying the response of Drosophila to viral infection we will uncover evolutionarily conserved mechanisms of innate immunity that, together with the programs developed in the other participating laboratories, will help to unravel the essential aspects of the antiviral response in mammals. The project has two specific aims, which are based on the original results obtained by the laboratory in the past five years. The picture emerging from these studies is that of a complex, multi-faceted program of defense, essentially based on RNA interference and an inducible response, on which our knowledge is still fragmentary. In this new application, we aim to produce an integrative view of the sensing, signaling and effector mechanisms leading to resistance to virus infection in drosophila. The first aim is to conduct unbiased forward genetic screens to identify mutations that impair the antiviral response. The readout for the screens will be the viral load (using GFP-expressing recombinant viruses), and the survival to viral infection. We propose to take advantage of the technological breakthrough of SOLID high throughput sequencing to rapidly identify the mutated genes in a cost effective manner by whole genome resequencing. The second aim is to characterize the role of three evolutionarily conserved pathways, namely RNAi, Toll and Imd, in antiviral defense in drosophila. The experimental strategy will be to identify by a powerful method combining the unparalleled affinity of avidin for biotin and the exquisite sensitivity of nanoLC-tandem mass spectrometry the molecules interacting with selected components of the RNA interference, Imd and Toll pathways. The composition of these complexes will be compared in cells either uninfected or infected by viruses replicating in different cytoplasmic locations. The function of the identified molecules will be characterized in cells and flies. In addition, we will study by live imaging in infected cells the interaction of the canonical molecules of the RNAi pathway (Dicer-2, R2D2 and AG02) with viral RNAs. RELEVANCE (See instructions): We anticipate that this project will allow us to reveal novel evolutionarily conserved pathways of host- defense, as we earlier did for Toll-like receptors. Understanding the molecular mechanisms of antiviral immunity in drosophila may also lead us to propose novel strategies to fight viral infections. Finally, this project is expected to shed light on the interaction between vector insects and the pathogenic arthropod- borne viruses they transmit to humans.